Oral Immunization with Attenuated Salmonella Choleraesuis Expressing the P42 and P97 Antigens Protects Mice against Mycoplasma hyopneumoniae Challenge

ABSTRACT Mycoplasma hyopneumoniae (M. hyopneumoniae, Mhp) is the etiological agent of swine enzootic pneumonia (EP), which has been associated with considerable economic losses due to reduced daily weight gain and feed efficiency. Adhesion to the cilia is important for Mhp to colonize the respiratory epithelium. Therefore, a successful vaccine must induce broad Mhp-specific immune responses at the mucosal surface. Recombinant attenuated Salmonella strains are believed to act as powerful live vaccine vectors that are able to elicit mucosal immune responses against various pathogens. To develop efficacious and inexpensive vaccines against Mhp, the immune responses and protection induced by recombinant attenuated Salmonella vaccines based on the P42 and P97 antigens of Mhp were evaluated. In general, the oral inoculation of recombinant rSC0016(pS-P42) or rSC0016(pS-P97) resulted in strong mucosal immunity, cell-mediated immunity, and humoral immunity, which was a mixed Th1/Th2-type response. In addition, the levels of specific IL-4 and IFN-γ in the immunized mice were increased, and the proliferation of lymphocytes was also enhanced, confirming the production of a good cellular immune response. Finally, both vaccine candidate strains were able to improve the weight loss of mice after a challenge and reduce clinical symptoms, lung pathological damage, and the inflammatory cell infiltration. These results suggest that the delivery of protective antigens with recombinant attenuated Salmonella vectors may be an effective means by which to combat Mhp infection. IMPORTANCE Mhp is the main pathogen of porcine enzootic pneumonia, a highly infectious and economically significant respiratory disease that affects pigs of all ages. As the target tissue of Mhp infections are the mucosal sites of the respiratory tract, the induction of protective immunity at the mucosal tissues is the most efficient strategy by which to block disease transmission. Because the stimulation of mucosal immune responses is efficient, Salmonella-vector oral vaccines are expected to be especially useful against mucosal-invading pathogens. In this study, we expressed the immunogenic proteins of P42 and P97 with the attenuated Salmonella Choleraesuis vector rSC0016, thereby generating a low-cost and more effective vaccine candidate against Mhp by inducing significant mucosal, humoral and cellular immunity. Furthermore, rSC0016(pS-P42) effectively prevents Mhp-induced weight loss and the pulmonary inflammation of mice. Because of the effectiveness of rSC0016(pS-P42) against Mhp infection in mice, this novel vaccine candidate strain shows great potential for its use in the pig breeding industry.

2. In figure 3, Antibody response induced by vacine candidates in mice. The question is why the author used log10 to deal with their data, I think log2 is more reasonable because the titer (double dilution) is adopted for statistical analysis. 3. In figure 4, Cell-mediated imune response and antigen-specif stimulation IFN-γ and IL-4 production. Panel DE seems unnecessary as ELISPOT has already proved that cell-dependent immunity. 4. In figure 6, Histolgicalnalysi and patholgical scores in the lung. Please show the ruler and magnification, a broad vision for this HE analysis is required to show a similar result. 5. The manuscript said that a live M. hyopneumoniae vaccine is available on the market, and the vaccine is not good, why did the author set up a control to compare with the current vaccine? I still believe that a live bacteria vaccine is better than a vectored vaccine delivering only one or two antigens.
Reviewer #2 (Comments for the Author): M. hyopneumoniae is a very important pathogen for pigs, which causes great lost to pig production world widely. However, currently used vaccines, especially inactivated vaccines, cannot provide satisfactory protection. Therefore, research on new vaccines is very important for this bacterium. This study reported that attenuated Salmonella Choleraesuis expressing P42 or P97 antigens protected mice against M. hyopneumoniae challenge. The results suggest P42 and P97 proteins as good candidates and the Salmonella Choleraesuis as a good vector to deliver antigen as live vector vaccine against M. hyopneumoniae. There are still some questions for the authors: 1. The mouse challenge model is not a common model used in the study of M. hyopneumoniae. The author needs to explain why mice were selected. Is there any literature basis? Why the IP and IN inoculation was chosen for challenge? In the discussion part, a paragraph needs to be added to discuss the differences and similarities of the pathogenicity between the mouse model and the pig model, including the possible differences between the vaccine evaluation results on the mouse model and the pig model. 2. Line 422-427: Was P97 full length protein or P97 fragment used in this study? It should be clear here. If it was a segment, specific information should be listed. 3. Did the author evaluate the in vivo growth of the attenuated Salmonella vaccines in mice after immunization? 4. The legend of Figure 3 is very confusing now. Which is the detection of anti-P42 antibody or anti-P97 antibody in Fig 3A? Why both pS-P42 and pS-P97 induced same antibody? All the samples should be detected with the same coating protein. The description of "weeks after immunization" in Fig 3A means "weeks after primary immunization" or "weeks after second immunization"? And, it did not clearly point out what is Fig 3D, 3E or 3F in the legend. 5. Line 474: how many animals were used for lymphocyte proliferation response test? The same questions also exist in line 452, how many animals were used for BALF samples collection. 6. Line 514: When was the animal autopsy performed? 21 days after infection? Please be clearly here. The time of autopsy is very critical for a challenge model. Why did the author choose this time point? Have the authors compared the pathological changes at different time points previously? 7. Line 518: How many fields of vision were scored for statistical analysis? 8. The lung lesions induced by M. hyopneumoniae varies among different lung lobes or positions. Was there a similar phenomenon in mice? If so, how to avoid these differences when carried out the pathological scoring? 9. Line 521-522. It should be clear what data was analyzed by Student's t test and what data was analyzed by one-way ANOVA.

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In this paper, the authors produced vaccines against Mycoplasma hyopneumoniae infection in mice utilizing attenuated Salmonella Choleraesuis vectors expressing immunogenic proteins P42 and P97. However, there are other concerns that require attention. 2. In recent years, P97 has been the most investigated adhesion factor. According to reports, M. hyopneumoniae adhesion to swine cilia is mediated by the R1 repeat region of the P97 adhesin. The paper does not specify which portion was expressed by the P97 plasmid.

It has been reported that attenuated Salmonella typhimurium expresses
3. Mycoplasma pneumoniae is typically transmitted to pigs through the intranasal route. Why did the authors administer the in vivo challenge by intraperitoneal and nasal drop? What is the rationale for intraperitoneal infection selection? What was the infectious dosage for each type?
The authors have merely indicated in the procedures that "10 8 CCU of the F66 strain will be administered intraperitoneally and nasally". 4. Absence of trials evaluating safety, as the author took off the sopB gene to minimize the intestinal inflammatory response of the host induced by the Salmonella vector itself. 5. The language must be edited by a native speaker or a professional translation agency. Please correct the inaccuracies such as INF.

Reviewer #1
On behalf of my co-authors, we would like to express our deep thanks to you for the constructive comments and suggestions, which are valuable in improving the quality of our manuscript. We have studied the reviewers' comments carefully and endeavored to revise and improve the manuscript accordingly. We would like to express our deep thanks for your helpful comments and hope that we have now produced a more balance and better account of our work. The main corrections in the paper and the detailed point-by-point responses to the reviewers' comments are listed below. We hope that the corrections will meet with your approval. We have marked the major changes with red in the revised manuscript.

Your comments:
In the issue of the proteins expression in Salmonella vaccine, if polyclonal antibody were used for detecting these two antigens expression, please show the data, I do not believe that "strains carrying the empty vector plasmids did not produce any protein that reacted withe polyconal antibody". Our response: Thank you for your valuable suggestions. We are sorry for the inaccuracies in the manuscripts. Based on your explanation, we recognize that the WB description in the last submission is improper. We have revised the manuscript according to your comments (Lane 163-165; Lane 797-799). At the same time, the WB results after polyclonal antibody incubation are also resubmitted, hoping to solve your confusion.

Your comments:
In figure 3, Antibody response induced by vacine candidates in mice. The question is why the author used log10 to deal with their data, I think log2 is more reasonable because the titer (double dilution) is adopted for statistical analysis. Our response: Thank you very much for your constructive comments. This point is very excellent. Based on your comments, we processed and analyzed the antibody titers using log2. At the same time, Fig. 3 has also been modified, which is believed to be more reasonable.

Your comments:
In figure 4, Cell-mediated imune response and antigen-specif stimulation IFN-γ and IL-4 production. Panel DE seems unnecessary as ELISPOT has already proved that cell-dependent immunity. Our response: Thanks for your valuable comments. To better demonstrate that the vaccine candidates in this study can induce cell-dependent immune responses, we conducted experiments from multiple perspectives. The ELISPOT experiment was mainly verified from the protein level, and the mRNA level of cytokines was a further verification of this result, which proved the reliability of the result.

Your comments:
In figure 6, Histolgicalnalysi and patholgical scores in the lung. Please show the ruler and magnification, a broad vision for this HE analysis is required to show a similar result. Our response: Thank you very much for your constructive comments. The comments were valuable and helpful for revising and improving our paper. Based on your comments, we have supplemented the magnifications and rulers of the pathological sections (Lane 832-833). At the same time, pathological sections with a broad vision are also provided, which are believed to be more helpful to prove the accuracy of our results (Fig. 6).

Your comments:
The manuscript said that a live M. hyopneumoniae vaccine is available on the market, and the vaccine is not good, why did the author set up a control to compare with the current vaccine? I still believe that a live bacteria vaccine is better than a vectored vaccine delivering only one or two antigens. Our response: Thank you for your comment. We fully agree with this view. Indeed, the 168 live vaccine strain (Mhp-168) is one of the Mhp vaccine strains that are used widely in China. And commercial attenuated vaccines based on Mhp-168 have been shown to be effective to decrease respiratory symptoms and increase growth parameters in commercial pig herds. Therefore, the study design would have been more reasonable and the results would have been more powerful if it had set a Mhp-168 control group. In follow-up studies to the current study, we will address this limitation by setting Mhp-168 control group. Like reviewer said, live vaccines are able to provide more protection than vaccines that only provide one or two antigens. Therefore, in follow-up studies, we will screen more antigens. At the same time, it will also be investigated whether the combined use of multiple antigens provides better protection.

Reviewer #2
On behalf of my co-authors, we would like to express our deep thanks to you for the constructive comments and suggestions, which are valuable in improving the quality of our manuscript. We have studied the reviewers' comments carefully and endeavored to revise and improve the manuscript accordingly. We would like to express our deep thanks for your helpful comments and hope that we have now produced a more balance and better account of our work. The main corrections in the paper and the detailed point-by-point responses to the reviewers' comments are listed below. We hope that the corrections will meet with your approval. We have marked the major changes with red in the revised manuscript.

Your comments:
The mouse challenge model is not a common model used in the study of M. hyopneumoniae. The author needs to explain why mice were selected. Is there any literature basis? Why the IP and IN inoculation was chosen for challenge? In the discussion part, a paragraph needs to be added to discuss the differences and similarities of the pathogenicity between the mouse model and the pig model, including the possible differences between the vaccine evaluation results on the mouse model and the pig model. Our response: Thank you for your comments. Indeed, as you said, mice are not a commonly used model for Mhp research. Conventional pigs have been the only animals widely used in pathogenicity and vaccine evaluation studies of Mhp for a long time (1-3). However, as an animal model, pigs are more expensive and have certain difficulties in operation. Therefore, in this study, we tried to use BALB/c mice with more advantages, such as inexpensive, smaller size, and easy to operate, as an animal model. Moreover, BALB/c mice is the most widely used animal model, and has been used for the pathogenesis and vaccine research of various zoonotic diseases. Moreover, the Jiangsu Academy of Agricultural Sciences in China has successfully established a mouse infection model of Mhp in 2017 and used it to identify the virulence of Mhp (Patent Application No.: CN201711362236.X; Announcement No.: CN108094315A). It is proved that mice can be used as a model of Mhp infection. The choice of the challenge route is because the patent of the Jiangsu Academy of Agricultural Sciences proves that the IP and IN routes can be used to infect mice with Mhp. Thanks for your suggestion, we have added differences in pathogenicity between mouse and pig models to the Discussion section, and also discussed possible differences in vaccine evaluation between the two models (Line 357-382).

Your comments:
Line 422-427: Was P97 full length protein or P97 fragment used in this study? It should be clear here. If it was a segment, specific information should be listed. Our response: Thank you for your valuable comments. We chose the C-terminal region (R1R2) of